Platelet adhesion at sites of vascular damage is required for normal haemostasis. Normally, platelets are prevented from activation in the blood stream by inhibitors of endothelial origin such as protacycline, nitric oxide or ectonucleotidase. However in some circumstances, dysfunctional endothelial cells loose their protective properties and promote platelet adhesion, platelet aggregation and growth of a solid thrombus on the site of the endothelial lesion.
Accordingly reliable methods for determining platelet susceptibility to activation in a patient are particularly desirable for diagnostic purposes.
Circulating platelets adhere to proteins of the subendothelial matrix exposed by an injured vessel in a process involving several receptors. Collagen fibers are highly thrombogenic and the platelet Glycoprotein (GP)VI predominantly mediates collagen-induced platelet responses.
GPVI is a platelet specific receptor of the immunoglobulin (Ig) superfamily containing two extracellular Ig domains (D1 and D2), a single transmembrane domain and a short cytoplasmic tail. GPVI shares with other receptors of the same family (e.g. FcαRI, TCR, and BCR) the particularity that the extracellular recognition (ligand-binding) domain and the intracellular signaling domain are located on separate subunits. GPVI signals through the non-covalently associated immune receptor adaptor FcRγ that presents an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The receptor is assembled via a transmembrane interaction between an Asp residue in the FcRγ homodimer and an Arg residue (R273) of GPVI. Upon stimulation, the Tyr residues of the ITAM are phosphorylated in an early and obligatory event triggering the signaling cascade and the mutation of R273 prevents both GPVI association with FcRγ and collagen induced signals.
There are growing evidence that optimal binding of GPVI to collagen depends on the formation of GPVI dimers at the platelet surface. Miura and coworkers (Miura, Y., et al., Analysis of the interaction of platelet collagen receptor glycoprotein VI (GPVI) with collagen. A dimeric form of GPVI, but not the monomeric form, shows affinity to fibrous collagen. J Biol Chem, 2002. 277(48): p. 46197-204), using recombinant proteins, were the first to report that collagen binds to the dimeric but not to the monomeric form of GPVI and that only the former was able to attenuate collagen-induced platelet aggregation. Crystallographic data showing dimerization of GPVI ectodomains (Herr, A. B., Direct evidence of a native GPVI dimer at the platelet surface. J Thromb Haemost, 2009. 7(8): p. 1344-6), studies using synthetic peptides with differentially spaced GPVI binding motifs to activate the receptor in platelets (Smethurst, P. A., et al., Structural basis for the platelet-collagen interaction: the smallest motif within collagen that recognizes and activates platelet Glycoprotein VI contains two glycine-proline-hydroxyproline triplets. J Biol Chem, 2007. 282(2): p. 1296-304) and studies using chemical cross linking agents (Berlanga, O., et al., Glycoprotein VI oligomerization in cell lines and platelets. J Thromb Haemost, 2007. 5(5): p. 1026-33) have strongly reinforced the notion that GPVI functions as a dimer. However, the valence of GPVI on resting and on activated platelets respectively is still a matter of debate.